Mayito Jonathan, Andia Biraro Irene, T Reece Stephen, R Martineau Adrian, P Kateete David
Department of Immunology and Molecular Biology, School of Biomedical Sciences, Makerere University College of Health Sciences, Kampala, +256, Uganda.
Department of Internal Medicine, School of Medicine, Makerere University College of Health Sciences, Kampala, +256, Uganda.
AAS Open Res. 2020 Jul 29;3:34. doi: 10.12688/aasopenres.13108.1. eCollection 2020.
: Tuberculin skin test and interferon gamma release assay (IGRA) show limitations in diagnosing latent tuberculosis infection (LTBI) and poorly predict progression to active tuberculosis. This study will explore detection of ( ) DNA in CD34 peripheral blood mononuclear cells (PBMCs) as a biomarker for LTBI and monitoring chemoprophylaxis response. In a cross-sectional study, 120 household contacts (60 HIV positive and 60 HIV negative) will be recruited. Also, 10 patients with sputum positive pulmonary tuberculosis and 10 visitors from low incidence countries with no history of TB treatment will be recruited as positive and negative controls, respectively. Participants will donate 100 ml (50 ml for TB patients) of blood to isolate PBMCs using density gradient centrifugation. Isolated PBMCs will be separated into CD34 and CD34 enriched cellular fractions. DNA from each fraction will be purified, quantified and subjected to droplet digital PCR targeting (a Complex multi-copy gene) and , a single copy gene. Also, 4 ml of blood will be drawn for IGRA. In a nested prospective study, 60 HIV positive participants will be given 300 mg of Isoniazid Preventive Therapy (IPT) daily for six months, after which they will donate a second 100 ml blood sample that will be processed as described above. Data from the cross-sectional study will be analysed to determine the proportion of individuals in whom DNA is detectable in CD34 and CD34 fractions and number of genomes present. Data from the prospective study will be analysed to compare the proportion of individuals with detectable DNA in CD34 and CD34 fractions, and median genome copy number, post vs pre-IPT. This study will determine whether detection of DNA in CD34 PBMCs holds promise as a biomarker for LTBI and monitoring chemoprophylaxis response.
结核菌素皮肤试验和干扰素γ释放试验（IGRA）在诊断潜伏性结核感染（LTBI）方面存在局限性，且对进展为活动性结核病的预测能力较差。本研究将探索检测CD34⁺外周血单个核细胞（PBMCs）中的（ ）DNA，作为LTBI的生物标志物并监测化学预防反应。在一项横断面研究中，将招募120名家庭接触者（60名HIV阳性和60名HIV阴性）。此外，将分别招募10例痰涂片阳性的肺结核患者和10名来自低发病率国家且无结核治疗史的访客作为阳性和阴性对照。参与者将捐献100毫升血液（肺结核患者为50毫升），通过密度梯度离心法分离PBMCs。分离出的PBMCs将被分为CD34⁺和CD34⁻富集细胞组分。每个组分的DNA将被纯化、定量，并进行针对（一个复杂的多拷贝基因）和（一个单拷贝基因）的液滴数字PCR。此外，将抽取4毫升血液进行IGRA检测。在一项嵌套的前瞻性研究中，60名HIV阳性参与者将每天服用300毫克异烟肼预防性治疗（IPT），持续6个月，之后他们将捐献第二份100毫升血液样本，按照上述方法进行处理。将分析横断面研究的数据，以确定在CD34⁺和CD34⁻组分中可检测到（ ）DNA的个体比例以及（ ）基因组的数量。将分析前瞻性研究的数据，以比较IPT前后CD34⁺和CD34⁻组分中可检测到（ ）DNA的个体比例以及（ ）基因组拷贝数中位数。本研究将确定检测CD34⁺ PBMCs中的（ ）DNA是否有望作为LTBI的生物标志物并监测化学预防反应。
